A successful immune response requires coordinated interaction of multiple cell types. The interaction between T-helper cells (Th) and antigen-presenting cells (APC) such as B cells, monocytes, and dendritic cells results from complex communications involving signals received through soluble cytokines or membrane-bound proteins as well as adhesive interactions. Many of these signals are not specific to a directed immune response and the proteins are broadly distributed.
A number of important T cell surface proteins involved in cell-cell interactions have been identified including CD2, CD4, CD8, CD28, LFA-1, CTLA-4 and gp39. These proteins participate in cell-cell contact by binding to their counter-receptors on APC and provide important costimulatory signals to T cells which modulate signals received through the T-cell antigen receptor. These costimulatory signals are necessary for the T cell to become fully engaged and express both membrane-bound and soluble factors required for the proper activation of the T cell-dependent effector cells (B cells, natural killer cells, monocytes, neutrophils, etc.). The gp39/CD40 T cell ligand/B cell receptor pair plays a critical role in the humoral immune response. In vitro studies have shown that this receptor/ligand pair is involved in B cell proliferation, antibody and cytokine production and cell viability. Studies in vivo, both through blocking with a monoclonal antibody or by observation of a genetic defect in gp39, have validated the in vitro results, and extended them to the requirement for a functional gp39 for germinal center formation during immune response to antigen.
CD40 is a 50 kDa type I membrane glycoprotein expressed by B cells, macrophages, follicular dendritic cells, thymic epithelium, normal basal epithelium, some carcinoma and melanoma-derived cell lines (Clark and Ledbetter 1986, Proc. Nat'l. Acad. Sci. USA 83:4494; Paerlie et al. 1985, Cancer Immunol. Immunother. 20:23, Ledbetter et al. 1987, J. Immunol. 138:788; Young et al. 1989, Int. J. Cancer 43:786; Galy and Spits 1992, J. Immunol. 149:775, Alderson et al. 1993, J. Exp. Med 178:669) and recently has been reported to be expressed on T cells (Armitage et al. 1993, Eur. J. Immunol. 23: 2326). It has been shown to be an important signaling molecule with a range of downstream effects in multiple systems. Early studies showed that CD40 was involved in B cell activation. Crosslinking CD40 with anti-CD40 monoclonal antibody induces B cell aggregation via LFA-1 (Gordon et al. 1988, J. Immunol. 140:1425, Barrett et al., 1991, J. Immunol. 146:1722), increases Ser/Thr (Gordon et al. 1988, supra) and Tyr (Uckun et al. 1991, J. Biol. Chem. 266:17478) phosphorylation of a number of intracellular substrates and provides a "competence" signal that allows B cells to proliferate and undergo class switching when stimulated with the appropriate second signal. For example, anti-CD40 monoclonal antibody can synergize with PMA (Gordon et al. 1987, Eur. J. Immunol. 17:1535) or anti-CD20 monoclonal antibody (Clark and Ledbetter 1986, supra) to induce B cell proliferation, with IL-4 to induce B cell proliferation (Gordon et al. 1987, supra; Rousset et al. 1991, J. Exp. Med. 172:705) and IgE secretion (Jabara et al. 1990, J. Exp. Med. 172:1861; Gascan et al. 1991, J. Immunol. 147:8; Rousset et al. 1991, supra; Zhang et al. 1991, J. Immunol. 146.1836, Shapira et al. 1992, J. Exp. Med. 175:289) and with IL-10 and TGF-.beta. to induce IgA secretion by sIgD.sup.+ B cells (DeFrance et al. 1992, J. Exp. Med. 175:671).
Isolation of a cDNA clone encoding human CD40 (Stamenkovic et al. 1989, EMBO J. 8:1403) shows that CD40 has a significant homology to the nerve growth factor receptor family. Using a soluble form of CD40, CD40-immunoglobulin fusion protein (CD40-Ig) (Armitage et al. 1992, Nature 357:80; Lane et al. 1992, Eur. J. Immunol. 22:2573; Noelle et al. 1992, Proc. Nat'l. Acad. Sci. USA 89:6550), it was found that the CD40 ligand (gp39, CD40-L), a protein of approximately 39 kDa, was expressed by activated human and murine T cells. In addition, blocking studies with CD40-Ig (Fanslow et al. 1992, J. Immunol. 149:655; Noelle et al. 1992, supra) or an anti-murine gp39 monoclonal antibody (MR1) Noelle et al. 1992, supra) showed that preventing gp39-CD40 binding resulted in inhibition of B cell biological responses.
Complementary DNA encoding both murine (Armitage et al. 1992, Nature 357:80) and human (Hollenbaugh et al. 1992, EMBO J. 11:4313; Spriggs et al. 1992, J. Exp. Med. 176:1543) gp39 or a soluble recombinant form of gp39 and IL-4 or gp39 and IL-10 can drive human B cells to secrete IgE and IgA, or IgG and IgM, respectively (Aruffo et al. 1993, Cell 72:291). Taken together, these results suggest that gp39 may be a T cell "switch" responsible for some aspects of B cell differentiation and isotype switching (Noelle et al. 1992, Immunol. Today 13:431).
Recently, the gene encoding gp39 was mapped to Xq26, the X chromosome region where the gene responsible for hyper-IgM syndrome (HIM) had previously been mapped (Aruffo et al. 1993, Cell 72:291). The gp39 molecules in the HIM patients were found to be functionally abnormal. Activated T cells have been found to produce normal levels of mRNA, but the gp39 encoded is defective (Aruffo et al. 1993, supra; DiSanto et al. 1993, Nature 361:541).
Hyper-IgM syndrome is one of at least seven inherited immunodeficiencies mapped to the X-chromosome (Kinnon and Levinsky 1992, J. Inherit. Metab Dis. 15:674). The disease is characterized by low or absent IgG, IgA and IgE levels, normal or elevated levels of IgM, normal numbers of recirculating B cells, susceptibility to bacterial and opportunistic infections (including Pneumocystic carinii), no germinal centers, autoimmunity, neutropenia, X-linked and autosomal forms, and gp39 ligand gene defects in the X-linked form of the disease. Common Variable Immunodeficiency (CVI) is another group of immunodeficiency disorders characterized by abnormal antibody responses and recurrent bacterial infections. Clinical presentations of CVI are diverse, as the disorders described by the term include a wide variety of as yet uncharacterized defects. Disease states described as CVI commonly show decreased or absent serum IgG and IgA, while the levels of IgM may be normal or decreased. Although most CVI patients have normal T cell numbers and responses, some may have decreased numbers, abnormal CD4/CD8 cell ratios or abnormal T cell function. There is also an increased probability of autoimmune antibodies in this patient population.
Mutations in the gene encoding gp39 result in deletions giving rise to frame shifts and premature stop codons, or point mutations resulting in amino acid substitutions (Allen et al. 1993, Science 259:990; DiSanto et al. 1993, supra; Fuleichan et al. 1993, supra, Korthauer et al. 1993, supra; Aruffo et al. 1993, supra; Collard et al. 1993, Immunol. Today 14:559). The effect of these mutations on expression of gp39 by activated T cells has been examined using soluble CD40-Ig, polyclonal antibody raised against a gp39 bacterial fusion protein (anti-TRAP) (Grafet al. 1992, Eur. J. Immunol 22:3191; Korthauer et al. 1993, Nature 361:539) and a gp39 specific monoclonal antibody 5c8 (Lederman et al. 1992, J. Exp. Med. 75:1091). Staining with soluble CD40-Ig, gp39 expression was found to be absent, while that for anti-TRAP was normal on T cells from one out of three patients tested, which was confirmed using the monoclonal antibody. These results show that expression of gp39 is variable in HIM patients and it has been suggested that further work is needed to determine whether the variation in surface expression of mutant forms of gp39 correlates with HIM disease severity. In the absence of a family history of X-HIM, the disease is difficult to distinguish from CVI. The methods currently used to identify a defect in gp39 as the causative agent in X-HIM include the sequencing of nucleotides comprising the gp39 gene from cDNA formed from mRNA isolated from in vitro activated lymphocytes that do not bind CD40, but do contain mRNA encoding gp39. This method has been used to show one patient diagnosed with CVI actually suffers from hyper IgM syndrome. However, the methods are laborious and would be very expensive to use on a more generalized basis.
What is needed in the art are additional monoclonal antibodies reactive with different epitopes of gp39 which can be easily used to assay for mutant forms of gp39 and for other purposes in diagnostics to distinguish between common variable immunodeficiency and X-linked hyper-IgM, and in therapeutic methods to modulate disease states responsive to interactions between CD40 and its ligand gp39.